The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants. / Science, Faculty of / Chemistry, Department of / Graduate

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression. / February 2008

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression.

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression.

Dry matter, crude protein and starch degradation characteristics of one corn (Pioneer 39P78) and four barley grain varieties (CDC Bold, CDC Dolly, Harrington and Valier) were evaluated in two in situ nylon bag trials. Trial 1 compared ground and rolled treatments of Harrington barley and Pioneer 39P78 corn, whereas Trial 2 evaluated ground and rolled treatments of the four barley varieties. Rumen degradability characteristics were compared with analytical results from thermal- and synchrotron-source FTIRM. Infrared absorbance spectra were collected from corn and four barley varieties using thermal-source FTIRM on the mid-IR beamline at the Canadian Light Source, Ltd. (Saskatoon, SK). Synchrotron-source FTIRM spectral data was collected for corn, Harrington barley and Valier barley on the U2B mid-IR beamline at NSLS-BNL (Upton, NY). CHO:Amide I peak area ratios were compared to the in situ rumen degradation results to determine if FTIRM spectral data could be related to the rate and extent of rumen degradation, and if thermal- and synchrotron-source FTIRM yielded different results. A grain x processing method interaction (P<0.01) was observed in both in situ trials where grinding produced a greater increase in the rate and extent of rumen degradation for Harrington barley than it did for corn (Trial 1) along with a greater increase in the rate and extent of rumen degradation for CDC Bold and CDC Dolly than for Harrington and Valier (Trial 2). Among barley varieties, increasing rate and extent of rumen degradation (CDC Bold>CDC Dolly>Harrington>Valier) corresponded to increasing starch:protein ratio as estimated by chemical analysis. This relationship was reversed for corn and Harrington barley where corn had a higher starch:protein ratio yet slower rumen degradation kinetics. For both thermal- and synchrotron-source FTIRM, CHO:amide I peak area ratios were greater (P<0.05) for corn than for Harrington barley. Comparison of CHO:amide I peak area ratios of barley varieties measured with thermal-source FTIR showed that varieties with higher (P<0.05) CHO:Amide I peak area ratios generally had higher rate and extent of rumen degradation. This indicates that starch:protein ratio estimated with FTIRM may be an indicator of rumen degradability characteristics when comparing varieties of the same grain, but not for different species of grains.

Dry matter, crude protein and starch degradation characteristics of one corn (Pioneer 39P78) and four barley grain varieties (CDC Bold, CDC Dolly, Harrington and Valier) were evaluated in two in situ nylon bag trials. Trial 1 compared ground and rolled treatments of Harrington barley and Pioneer 39P78 corn, whereas Trial 2 evaluated ground and rolled treatments of the four barley varieties. Rumen degradability characteristics were compared with analytical results from thermal- and synchrotron-source FTIRM. Infrared absorbance spectra were collected from corn and four barley varieties using thermal-source FTIRM on the mid-IR beamline at the Canadian Light Source, Ltd. (Saskatoon, SK). Synchrotron-source FTIRM spectral data was collected for corn, Harrington barley and Valier barley on the U2B mid-IR beamline at NSLS-BNL (Upton, NY). CHO:Amide I peak area ratios were compared to the in situ rumen degradation results to determine if FTIRM spectral data could be related to the rate and extent of rumen degradation, and if thermal- and synchrotron-source FTIRM yielded different results. A grain x processing method interaction (P<0.01) was observed in both in situ trials where grinding produced a greater increase in the rate and extent of rumen degradation for Harrington barley than it did for corn (Trial 1) along with a greater increase in the rate and extent of rumen degradation for CDC Bold and CDC Dolly than for Harrington and Valier (Trial 2). Among barley varieties, increasing rate and extent of rumen degradation (CDC Bold>CDC Dolly>Harrington>Valier) corresponded to increasing starch:protein ratio as estimated by chemical analysis. This relationship was reversed for corn and Harrington barley where corn had a higher starch:protein ratio yet slower rumen degradation kinetics. For both thermal- and synchrotron-source FTIRM, CHO:amide I peak area ratios were greater (P<0.05) for corn than for Harrington barley. Comparison of CHO:amide I peak area ratios of barley varieties measured with thermal-source FTIR showed that varieties with higher (P<0.05) CHO:Amide I peak area ratios generally had higher rate and extent of rumen degradation. This indicates that starch:protein ratio estimated with FTIRM may be an indicator of rumen degradability characteristics when comparing varieties of the same grain, but not for different species of grains.